Evaluation of CD44 and TGF-B Expression in Oral Carcinogenesis

Statement of the Problem: Oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral cavity. Early diagnosis of OSCC by using biomarkers provides preventive treatment approach to suppress the disease in early stages. CD44 as a cancer stem cell (CSC) marker may be cleaved by MT1-MMP and plays an important role in migration of cancer cells. TGF-B promotes formation of invasive cancer cells phenotype through epithelial mesenchymal transition (EMT) and induces MT1-MMP formation. Purpose: The aim of this study was to evaluate the expression of TGF-B and CD44 in leukoplakia (premalignant lesion), squamous cell carcinoma (SCC), and normal oral mucosa to determine the role of these markers in the carcinogenesis process of the oral mucosa. Materials and Method: In this retrospective study, the expression of TGF-B and CD44 were evaluated in 55 paraffin-embedded specimens (10normal mucosa, 15 non-dysplastic leukoplakia, 15 dysplastic leukoplakia, and 15 OSCC) by immunohistochemistry. Statistical analyses were performed using Kruskal-Wallis, Mann-Whitney, and Spearman’s rank correlation tests. Results: Evaluation of CD44 and TGF-B expression in the four studied groups showed statistical significant difference for each marker (p< 0.001). Pairwise comparison of CD44 and TGF-B expression in all groups except normal mucosa and non-dysplastic leukoplakia demonstrated statistical significant difference. In addition, there was positive significant correlation between two markers (r= 0.914, p< 0.001). Diagnostic test’s accuracy for identification of OSCC and dysplastic leukoplakia from non-dysplastic leukoplakia and normal tissues and recognition of OSCC from dysplastic leukoplakia showed optimum sensitivity and specificity. Conclusion: Increased expression of CD44 as a cancer stem cell marker and TGF-B as an EMT marker from normal mucosa to non-dysplastic leukoplakia, dysplastic leukoplakia, and OSCC and also the significant correlation between these two markers indicated their role in carcinogenesis of oral mucosa.


Introduction
Squamous cell carcinoma (SCC) is the most common malignancy of the oral cavity. It accounts for more than 90% of all oral malignancies [1]. It has a morbidity and mortality rate of around 50% [2]. Despite the advances in novel therapeutic modalities, the mortality and mor-bidity rate of oral squamous cell carcinoma (OSCC) has not been significantly decreased in the recent years [3].
Epithelial carcinogenesis is a multi-phase process [4]. It is believed that initial changes occur in normal oral epithelium, which causes progression to oral dysplasia and cancer due to several genetic mutations. Oral carcinoma is the final outcome of this multi-step process that often occurs prior to morphological changes of the epithelium. Therefore, morphological changes do not always predict the possible progression of dysplasia to cancer.
Several multi-functional factors may play a role in progression and transformation of potentially malignant oral lesions to OSCC [5].
In recent years, various markers related to cancer stem cells (CSCs) have been identified, which affect the initiation, progression, and treatment resistance of cancers. CD44 is a cell surface glycoprotein and, as a CSC marker, plays an important role in cell migration and adhesion, tumor invasion, prognosis, and metastasis of cancers [2,6].
Epithelial mesenchymal transition (EMT) is an important factor for the initiation of invasive cancer cell phenotype and transition to carcinoma. Transforming growth factor-beta (TGF-B) is among the most important cytokines that induce EMT. TGF-B belongs to superfamily consist of more than thirty proteins including growth factors, bone morphogenetic proteins, and activins. TGF-B as a multi-functional cytokine plays an important role in proliferation, differentiation, and migration of cancer cells as well as cancer progression and metastasis of cancers [7][8][9].
Recently, it has been shown that CD44 is expressed in cancer cells and may be cleaved at the ectodomain by membrane type 1-matrix metalloproteinase (MT1-MMP), forming soluble CD44. Thus, it plays a critical role in cancer cell migration. TGF-B is among the markers that can induce MT1-MMP [10]. Therefore, further investigations on molecular diagnostic biomarkers including EMT and CSC markers may provide useful information about detection of malignant transformation.
To the best of our knowledge, this study is the first to assess simultaneous expression of these two markers in leukoplakia as a potentially malignant oral lesion.
The aim of this study was to investigate the expression of TGF-B as an EMT inducing marker and CD44 as a CSC marker in the process of carcinogenesis in oral mucosa.

Patients
This retrospective study evaluated 55 specimens inclu- Comparison of CD44 expression in the studied groups showed that the greatest mean total score was in OSCC group (3.87) and the lowest was in normal mucosa group (0.7). In addition, non-dysplastic and dysplastic leukoplakia lesions showed mean total score of 1.0 and 2.20, respectively (Table 1, Figure 1).  According to the Kruskal-Wallis test, a significant difference was observed among the groups (p< 0.001).
Pairwise comparison of CD44 expression in the studied groups was performed. All differences were found to be significant (p< 0.001) except comparison of nondysplastic leukoplakia and normal mucosa (p= 0.285).
Comparison of TGF-B expression among the four groups was performed according to the mean total score. OSCC lesions showed greatest mean total score   Discussion OSCC is the most prevalent oral cancer [1]. Diagnosis of OSCC in the preliminary phase by using biomarkers provides preventive treatment approach to suppress the disease in early stages. Thus, this study aimed to assess the expression of CD44 and TGF-B in OSCC and leukoplakia as a premalignant oral lesion.
Recent evidence has shown that only small subgroups of cells, referred as CSCs, with self-renewal capacity are responsible for tumor initiation and progression [13][14]. Thus, tumors are maintained by their own stem cells also known as self-sustaining cells [15][16].
A number of studies [3,6,17]  cell-cell and cell-matrix interaction as well as cell migration and tumor progression. It should be noted that EMT process has been the focus of various studies [6,9,14,18]. During the process of EMT, epithelial cells down-regulate the expression of cell-adhesion proteins and gain migratory and invasive properties. EMT is essential for numerous physiological processes such as fetus development, wound healing, fibrosis, progression of carcinoma, and metastasis [14,[18][19]. A correlation has been found between the EMT and invasive behavior of different types of cancers including colon cancer, head and neck squamous cell carcinoma and so on [6][7]18]. It has been reported that EMT plays a fundamental role in development of a malignant phenotype [19].
TGF-B is one of the most important cytokines that induces EMT. The EMT process is a topic of interest in cancer research due to its correlation with progression of different tumor types. In the recent years, the relationship of EMT with CSCs is an important aspect of various studies [14,17,19].
This study aimed to assess the expression of TGF-B as an EMT-inducing marker and CD44 as a CSC marker in the process of carcinogenesis in oral mucosa and the correlation between these two markers.
In the present study, the expression of CD44 was cancers [20][21].
It should be noted that according to the current results, CD44 had maximum sensitivity and specificity for differentiation of "SCC and dysplastic leukoplakia" from "non-dysplastic leukoplakia and normal mucosa" as well as differentiation of SCC from dysplastic leukoplakia. This finding indicated the correlation between the expression of this cell adhesion molecule as a CSC antigen and transition to OSCC.
Similar to the present study, the results of Abdulmajeed et al. [22], Rautava et al. [23], Oliveira et al. [2], and Bahar et al. [24], confirmed that increased expression of CD44 would increase the risk of malignancy.

Conclusion
Increased expression of CD44 as a CSC marker and TGF-B as a marker involved in EMT from normal mucosa to non-dysplastic leukoplakia, dysplastic leu-koplakia and OSCC and the significant correlation between these two markers indicated their role in carcinogenesis of oral mucosa. The markers evaluated in this study showed maximum sensitivity, specificity, and diagnostic accuracy for differentiation of "OSCC and dysplastic leukoplakia" from "non-dysplastic leukoplakia and normal mucosa" and also for differentiation of OSCC from dysplastic leukoplakia.